Enzymes

1. Enzymes Ms. Rahinaz Department of Biochemistry, Yenepoya Medical College, Yenepoya.

2. Enzymes – Table of Contents Introduction Classification Mechanism of Action Part I Enzyme Kinetics Enzyme Inhibition Enzyme regulation Part II Isoenzymes Clinical Applications of Enzymes 05/74

3. Table of contents – Part I in detail • Introduction, definition • Enzyme Vs Catalyst • Classification – IUB • Cofactor & Coenzyme • Metallo enzymes • Active site • Enzyme specificity • Mode(Mechanism) of Action of Enzymes • Theories –Michaelis–Menton Theory • Fisher’s Template Theory • Koshland’s Induced fit Theory • Enzyme Kinetics • Factors affecting Velocity – Km Value Significance 06/74

4. Background • Rate of a Reaction, Reversible & Irreversible Reaction and Reaction Equilibrium • Consider a chemical reaction, r1 (k1) • A+B C+D, where A & B are reactants, C & D are products, r1 is the rate (velocity or speed) of the reaction and k1 is the rate constant. • Since the rate of a reaction is directly proportional to the product of the concentrations of the reactants, • r1  [A] [B]

5. Therefore, • r1 = k1 [A] [B], where k1 is a rate constant. • Now consider a reversible reaction, r1 (k1) • A+B C+D r2(k2) • At the start, since there will be only the reactants and no products, the rate of forward reaction (left to right) will be maximum and that of backward reaction (right to left) is zero. • As the reaction proceeds, the concentrations of A and B decrease and those of C and D increase;

6. and therefore, the rate of forward reaction decreases and that of backward reaction goes on increasing. • After sometime a stage will be reached when the rate of the forward reaction becomes equal to the rate of backward reaction. Then the system is said to have attained a state of equilibrium – chemical equilibrium. • Hence the concentrations of all the reactants and products at equilibrium will have become stationary. • That is, at equilibrium, r1 = r2 • Since r1 = k1 [A] [B] and r2 = k2 [C] [D], • At equilibrium, k1 [A] [B] = k2 [C] [D] [A] [B] = k2 [C] [D] k1

7. [A] [B] = Keq (equilibrium constant) – law of • [C] [D] mass action, for reversible reactions • In a freely reversiblereaction, the value of Keq is 1 and there is no energy change (ΔG = 0); • In an irreversible reaction the Keq value is very high (endergonic reaction; ΔG = +ve) or negligible(exergonic reaction; ΔG = –ve).

8. A Catalyst • For example: Consider hydrolysis of sucrose: • Sucrose + H2O H+ glucose + fructose. HCI • The role of HCl here is “ Catalyst” • Accelerates the rate of reaction many folds.

9. CATALYSIS • A catalyst increasesthe rate of a chemical reaction but remains unchanged chemically at the end of the reaction. The phenomenon is catalysis.  Catalysts greatly enhance the rate of achievement of reaction equilibrium. • But they neither cause chemical reactions to take place nor change the equilibrium constant of chemical reactions. • Catalysts catalyze the forward and backward reactions equally. • A catalyst will only catalyze the reaction in the thermodynamically allowed direction.

10. Activation Energy • Energy required for a reaction to take place at room temperature is the activation energy. • Catalysts accelerate chemical reactions by lowering the activation energy or energy barrier for a reaction to take place. • When a reactant acquires enough energy (activation energy) to undergo transition to form the product its energy status is said to be in transition state (T). • Transition state represents the highest point in their energy barrier • Thus, it can also be said that a catalyzed reaction needs less energy to move to transition state.

11. Mechanism • For a reaction to take place, reactant molecules must collide with each other with sufficient energy. • Thus each reaction is said to have an energy barrier. • Eg: Hydrolysis of Sucrose • Sucrose + H2O + 290 Kcal Glucose + Fructose (energy barrier) • The minimum amount of energy that the reactants must possess to overcome this barrier is called “activation energy”

14. This Process is called “tunnelling through energy barrier” by providing alternate pathways. • Enzymes act by lowering the activation energy by the same mechanism. • Most of the reactions in the body have very high energy barriers, but enzymes accomplish these reactions at body temp, by lowering activation energy. Recall: • Eg: Sucrose + H2O+ 90 Kcal Sucrase Glucose + Fructose

17. Introduction • Enzymes are defined as biological catalysts which are protein in nature. • (Ribozyme is a biological catalyst made of RNA) • Like all proteins, enzymes are synthesized by living cells; active both inside and outside the cell and even in cell free extract, colloidal in nature and heat labile. • Compared to inorganic catalysts, enzymes are more specific, more efficient, larger in size and less stable. 07/74

18. Compared to inorganic catalysts…… Enzymes are more • Specific • More efficient • larger in size and • less stable.

19. General features of enzymes • There are millions of chemical reactions taking place in the body and all of them, except a few, are enzyme-catalyzed. • Very few reactions that are not enzyme catalyzed are called spontaneous or non-enzymatic reactions. •  The reactant/s on which the enzymes act to catalyze the reaction are called the substrate/s of the enzyme. • Enzymes are much larger than the substrates they act on.

20. General features ……..continued • Enzymes have active sitesoractive centers, where the catalysis takes place. • Active site is only a small portion of the enzyme. • The active site contains substrate binding site and catalytic site. •  Enzymes are huge in size but with small active sites.

21. Clinical Importance of Enzymes • Enzymes play central role in health & Diseases (Eg: Inherited genetic diseases due to deficiency of enzyme). Any disease has an enzyme background. • Many drugs exert their action through enzymes. • Measurement of enzymes in blood is useful for diagnosis and follow up. • Therapeutic importance

22. Biological importance of enzymes • Enzymes catalyse multiple dynamic processes, which make life possible. Eg: Digestion & absorption, breakdown of food to give energy, muscle contraction, synthesis & maintenance of tissues, fighting against infection, etc. • (Commercial importance of enzymes is in industrial applications)

23. Nomenclature (Trivial Names) • Enzymes are named by adding suffix –‘ase’ to the substrate • Ex: Lactase, sucrase, proteases • Nature of reaction • Ex: Dehydrogenase, transferase • Many enzymes are named after the reaction they catalyze.

24. Name of the substrate and nature of reaction they catalyse- • Ex: Pyruvate dehydrogenase, Glutathione reductase, Phospho hexose isomerase and Xanthin oxidase

25. Classification of enzymes • In 1964, IUB suggested a classification for enzymes which now widely accepted. • It is based on reaction type • As per this system enzymes are classified into 6 major classes, each of which is further sub divided into Sub class, Sub-sub class etc. • Each enzyme is given Enzyme – Code (EC) which is a 4 digit number. • Eg: Alcohol dehydrogenase EC is 1.1.1.1

26. Enzyme Classification 1. Oxido-reductases - Redox reaction. Transfer of hydrogen or oxygen or electrons) 1. Alcohol Dehydrogenase Eg: CH3OH HCHO Alcohol Aldehyde NAD NADH + H+ 2. Lactate dehydrogenase Pyruvate Lactate NAD NADH + H+ Oxido-reductases are further sub classified into oxidases, aerobic dehydrogenases, anerobic dehydrogenses, hydroperoxidases and oxygenases.

27. 2. Transferases • Transfer of a group(other than hydrogen) from one substrate to another. • Glucose Hexokinase Glucose – 6- Phosphate ATP ADP Alaninetransaminase • PyruvateAlanine Glutamate αKetoglutarate

28. 3. Hydrolases • Hydrolysis(Splitting of an anhydride bond with addition of water, like ester, peptide glycosidic bonds). Eg: All GIT enzymes Lactose Lactase Glucose + Galactose Sucrose sucrase Glucose + Fructose

29. 4. Lyases • Breaking of bond by other than hydrolysis. • Aldolase 1.Eg. Fructose -1,6 Bis- Phosphate Glyceraldehyde -3- • Phosphate DHAP • 2. Malate Fumarase Fumarate + Water

30. 5. Isomerases Isomerization ( Intramolecular rearrangement of atoms) 1 Eg: Phosphohexose isomerase Glucose – 6- Phosphate Fructose -6-Phoshate 2. Eg: Phosphotriose isomerase Glyceraldhyde -3- Phosphate DHAP Names of isomerases end with isomerases, epimerases, racemases or mutases.

31. 6. Ligases • Joining two molecules by covalent bond at the expense of energy (ATP). They are synthetases. • ATP ADP + Pi • 1. Eg : Pyruvate + CO2 Oxalo acetate Pyruvatecarboxylase ATP ADP + Pi 2. Glutamic Acid + NH3 Glutamine Glutamine synthetase Pneumonic – OTHLIL (Oh Thank Heaven Learning Is Lively)

32. Cofactors • Non protein factors required for enzyme action. • These are small molecules, heat stable. They may be: 1) Organic molecules – called Coenzymes 2) Inorganic molecules – called activators, usually metal ions like Zn2+ or Fe2+, Cl- , Mg 2+ Without these cofactors, the enzyme does not exhibit any catalytic activity.

33. Cofactors Organic molecules -Coenzymes -Prosthetic groups Inorganic molecules -Activators - Activators

34. Cofactors……. continued Some enzymes also contain a non protein part which is tightly bound to the enzyme called prosthetic group. • Ex: Haem in Cytochromes • Protein part of the enzyme is called apoenzyme Apoenzyme + Cofactor or prosthetic group Holoenzyme

35. Coenzymes • Non protein organic cofactors required for enzyme action • Small molecules, dialysable, heat stable. • Many Coenzymes, like substrates, bind reversibly by non covalant bonds to the enzyme active site. • They undergo chemical change during reaction, regenerated & released at the end of the reaction. Hence they can be considered as co- substrates.

36. Coenzymes .....continued Exception: Coenzymes FMN &FAD, Biotin, PLP are bound tightly to the enzyme by covalent bonds. • They are not specific to the enzyme (Enzyme has a specific coenzyme but a coenzyme can have many enzymes) (Ex: NAD has around 700 enzymes) • Hydrolases do not require coenzymes • Many coenzymes are vitamin derivatives. • Functions:Coenzymes act as carriers of various groups during the reaction (Addition or removal of a group from substrate to form the product):

37. Examples of Coenzymes

38. Clinical Importance of coenzymes • Since many of the coenzymes are vitamin derived, deficiency of specific vitamin leads to specific coenzyme deficiency resulting in diseases. Eg: Pellegra, Megaloblasticanemia, etc. Inorganic Cofactors (Activators) These are metal ions like Zn, Mg, Fe. They are of 2 Types: • Metallo enzymes– Metal ions requiring enzymes where metal is bound tightly to enzyme. Eg: Carbonic anhydrase (Zinc). • Metal Activated enzymes-Metal is not tightly bound to the enzyme. Eg: Enolase & ATPase (Mg 2+) • Cl- (Non Metal) – Activator of salivary amylase

39. Active site of the enzyme • Enzymes are big molecules compared to substrates. • Active site of an enzyme is a small region of the enzyme where substrate binding & subsequent catalysis takes place.

40. Active Site

42. Salient features of Active Site: • Situated in a pocket or cleft of the enzyme molecule. • It contains substrate binding site & catalytic site. • Specific substrate binds at the Substrate binding site reversibly by non covalent forces. • Contains specific groups or atoms which facilitate the binding of correct substrate.

43. Salient features of active site............. continued • Catalytic Site is constituted by one or more amino acid residues called as catalytic residues which are involved in catalysis.( Ser.,Thr etc are commonly found). • Cofactors required for some enzymes are also present as a part of the catalytic site.

44. Significance of Active Site : • Specificity of enzyme resides in active site. • Active Site is not a rigid structure but flexible; having 3- D structure. • When correct substrate binds, there is a conformational change in the structure of active site to hold the substrate tightly & subsequent catalysis. • Many drugs & poisons bind to active site of enzyme & bring about change in the enzyme activity.

45. Enzyme Specificity • Enzymes are highly specific to the reaction they catalyze. • Specificity of enzyme resides in active site because its shape is complementary to the substrate. • More specific when compared to an inorganic catalyst. • Types of Specificity 1. Absolute substrate specificity • 2. Broad substrate specificity

46. Absolute substrate specificity: Here enzymes catalyze only one reaction • Glucokinase to form Glucose -6 – Phosphate • Urease to form Ammonia + CO2 • L-Amino acid oxidase – does not act on D-amino acids • Lactase to form glucose and galactose

47. Broad substrate specificity: Enzymes can catalyze more than one reaction Eg: Hexokinaseacts on Hexoses (glucose, fructose, galactose). Proteases– trypsin, pepsin act on all proteins Aminopeptidases remove amino terminal end of all proteins.

48. Mechanism of enzyme action • The lowering of activation energy is explained by taking Michaelis –Menton model of enzyme catalyzed reaction. • As per this theory, enzyme(E) combines with substrate(S) to form enzyme – substrate complex(ES) which immediately dissociates to form product(P) releasing the free enzyme. E + S ES E + P (transition state)

50. Theories to explain formation of ES & subsequent catalysis: 1. Fischer’s template theory (Lock & Key model) 2. Koshland’s induced fit theory. • Template theory • As per this theory, the active site of the enzyme is the template which is rigid & complimentary to the • substrate. • It is just like correct key(substrate) which fits into the Lock(active site).